ES2260794T3 - STIMULATION OF GUEST DEFENSE MECHANISMS AGAINST TUMORS. - Google Patents

Abstract

A PROCEDURE TO TREAT NEOPLASTIC DISEASE IN A MAMMER, BY ADMINISTRATION TO THE MAMMER OF A THERAPEUTICALLY EFFECTIVE AMOUNT OF AN INTERFERON, BY NASOMUCOUS CONTACT. THE AMOUNT OF INTERFERON ADMINISTERED IS LESS THAN A QUANTITY THAT INDUCES A PATHOLOGICAL RESPONSE WHEN IT IS PARENTERALLY ADMINISTERED.

Description

Stimulation of the defense mechanisms of the host against tumors.

The present invention relates to procedures to stimulate host defense mechanisms against pathological diseases in a host mammal by means of administration of interferon through the oromucosa. In particular, The invention can be applied to methods for treatment of cancer

Background of the invention

Alpha interferons are widely used for the treatment of a large number of hematologic malignancies, including hair cell leukemia, chronic myelogenous leukemia, low-grade lymphomas, cutaneous T-cell lymphomas and solid tumors, such as by example renal cell carcinoma, melanoma, carcinoid tumors and AIDS-associated Kaposi sarcoma (Gutterman, JU, Proc. Natl. Acad. Sci. USA , 1994, 91 : 1198-1205; WO 8803411). Antitumor effects are usually observed at high doses, often on the order of tens of millions of units of interferon-? (IFN-?), Administered by parenteral injection. Interferon-? Is a Type I interferon, a class that also includes? And? Omega interferons. Although various routes of administration have been routinely used for the administration of type I interferons, including intravenous, subcutaneous, intramuscular, topical and intralesional injection, the oral route has not been used in general because interferons are proteins that are considered to be they are inactivated by proteolytic enzymes and are not absorbed appreciably in their native form in the gastrointestinal tract. In fact, several studies have been unable to detect blood interferons after oral administration (Cantell and Pyhäla, J. Gen. Virol ., 1973, 20 : 97-104; Wills et al ., J. IFN Res ., 1984 , 4 : 399-409; Gilson et al ., J. IFN Res ., 1985, 5 : 403-408).

There have been several anecdotal reports on the efficacy of low doses of interferon administered as a spray nasal or as an oral liquid formulation in the treatment of a diversity of diseases, particularly influenza. In a series of patent documents the use of doses has been described Interferon losses of origin of heterologous species administered orally for the treatment of infectious rhinotracheitis ("boarding fever") in cattle and feline leukemia and also for the treatment of other diseases, for the increase of the efficacy of vaccines; to improve the effectiveness of the food utilization; and for the prevention of theileriosis bovine See US Patent No. 4,462,985, the Australian patent . 608519, Australian Patent No. 583332 and U.S. Patent No. 5,215,741, respectively. Additionally, US Patent No. 5,017,371 describes the use of interferon in this way to the treatment of chemotherapy side effects or cancer radiotherapy In these documents, interferon used was human interferon-? prepared through the Cantell procedure, administered in a solution phosphate buffered saline at a dose of 0.41 to 5 IU per pound of body weight. Although said documents suggest that said doses Interferon losses administered in the oropharyngeal mucosa, preferably in a form adapted for prolonged contact with the oral mucosa, they can be effective for the treatment of a wide variety of diseases, including cancer, evidence experimental in the case of diseases other than fever boarding, feline leukemia, canine parvovirus and Theileriosis is very anecdotal. In particular, no adequate controlled trial of such treatment in any model Animal for human cancers.

Instead, the invention described herein document is based on the first controlled study in a model efficacy animal of interferon administered via oromucosal route for the treatment of cancer.

Summary Description of the Invention

The present invention provides a procedure for the treatment of cancer in a human through of administration to the human of a therapeutically amount Effective of an interferon through oromucosal contact. The amount of interferon administered is less than the amount that induces a pathological response when administered by route parenteral In particular, the invention provides a method for the treatment of multiple myeloma, cell leukemia hairy, chronic myelogenous leukemia, low grade lymphoma, cutaneous T-cell lymphoma, carcinoid tumors, cancer cervical, sarcomas, including Kaposi's sarcoma, tumors kidney, carcinomas, including cell carcinoma renal, hepatic cell carcinoma, nasopharyngeal carcinoma, hematological malignancies, colorectal cancer, glioblastoma, laryngeal papillomas, lung cancer, colon cancer, malignant melanoma or brain tumors, including malignant brain tumors. In an embodiment of the invention, the procedure can be applied in general for the Treatment of tumors of non-viral etiology.

Oromucosal administration may involve administer an effective dose of interferon in a single dose or the effective dose can be administered in several smaller doses for a period of time sufficient to obtain a stimulation of the host defense system equivalent to that of a single dose Also, the effective dose of interferon can be administered continuously for a period of time enough to get a stimulation of the defense system of the host equivalent to that of a single dose.

The procedure is put into practice. administering from about 1 x 10 4 IU to about 20 x 10 6 IU of interferon, more preferably from about 1 x 10 4 IU to about 1 x 10 6 IU of interferon, as long as the selected dose be such that it does not induce a parenteral pathological response, such and as defined in the present invention, or that is less than the amount that induces a pathological response if administered by parenteral route These dose ranges generally refer to α-homologous interferon in man. For another type of interferons, the dose that will induce a pathological response can differ from that induced by the α-homologous interferon in the man. A doctor treating a patient with a particular type of interferon will be able to easily identify the interval of adequate dose for the patient to be treated.

A pharmaceutical composition for your Oromucosal administration may comprise an amount therapeutically effective of at least one interferon. The Composition can be provided as a solution, compressed, tablet, gel, syrup, paste or by a delivery system Oromucosal controlled release. Optionally, the composition may contain buffers, stabilizers, thickening agents, absorption enhancers, viscosity enhancers and Similar.

The pharmaceutical composition can be provided in a dosage unit form having 1 x 10 4 IU at 20 x 10 6 IU of interferon, more preferably 1 x 10 4 IU at 1 x 10 6 IU of interferon.

The procedure can be implemented as the only therapeutic approach or as an adjuvant to a chemotherapy or radiotherapy or with other cytokines, such as interleukin-2, 12 or 15 or with inductors of IFN

The procedure is carried out using a Type I or II interferon, selected from α, β, γ, Ω and consensus interferons, more preferably with a recombinant IFN-?

Detailed description of the invention

The invention is described below in detail for reference only using the following Definitions and examples. All patents and publications at which are referred to in this document have been expressly incorporated as a reference.

Definitions

As used in the present invention, "interferon" refers to a Type I or Type II interferon, including those commonly referred to as α, β, γ, and mixtures thereof, including The consensus sequence. Interferons can be purchased from a wide variety of commercial sources and have been approved for the treatment of numerous indications. Interferon can be from natural sources, but is preferably a recombinant product. For the purposes of the invention, the term "interferon" also includes polypeptide fragments that have interferon activity and the chimeric or mutant forms of interferon in which modifications have been made to the sequence, for example, to increase stability, without affect the nature of its biological activity, as described in US Pat. 5,582,824, 5,593,667 and 5,594,107, between
others.

Optionally, interferon can be administered at the same time as an inducer of interferon synthesis and release. The inductor can be administered together with interferon or it can be administered separately. Interferon inducers include, for example, polynucleotides type poly I: C; an orally administrable interferon of low molecular weight is preferably used. Some suitable inductors have been described in the state of the art, for example Tilorone (US Patent No. 3592819; Albrecht et al ., J. Med. Chem ., 1974, 17 : 1150-1156) and the quinolone derivative Imiquimod (Savage et al ., Brit. J. Cancer , 1996, 74 : 1482-1486).

The procedures and compositions of the invention may optionally be used in conjunction with one or more different treatments for the specific disease, and the Assistant doctor or veterinarian will be able to easily select said other treatments that are appropriate according to circumstances.

In one embodiment, the invention provides a procedure for the treatment of cancer in a human, which it comprises the step of administration of interferon as it has been previously described. Cancer can be a cancer in metastasis.

Although the process of the invention may be used without simultaneous treatment with other agents, it contemplates that this embodiment of the invention will be particularly Useful in the following frames:

to)

how adjuvant therapy, after surgery, chemotherapy or radiotherapy performed using standard protocols

b)

for the treatment of interferon-sensitive cancers, the procedure of the invention is used in isolation or in conjunction with conventional chemotherapy or radiotherapy; Y

C)

for the treatment of interferon-resistant cancers, the procedure of the invention is used in isolation or more preferably in conjunction with chemotherapy or radiotherapy conventional.

The procedures indicated above are direct to the induction and / or maintenance of the remission of the disease. By "together with another treatment" is understand that interferon is given before, during and / or after of radiotherapy or other chemotherapy. The most appropriate protocol It will depend on various factors, as described later.

In particular, it is contemplated that the procedure of the invention will preferably be used in conjunction with as minimum a different treatment selected from the group that It consists of chemotherapy using cytostatic drugs, one or more different cytokines that have activity anti-cancer but have a mechanism of action different from interferon, anti-angiogenic agents and agents that enhance interferon activity. Preferably, the second cytokine is interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-12 (IL-12), or interleukin-15 (IL-15); preferably the angiogenesis inhibitor is AGM-1470 [ester (3R- (3α, 4α (2R *, 3R *), 5β, 6β)) - 5-methoxy-4- (2-methyl-3- (3-methoxy-2-butenyl) oxyranyl) -1-oxaspiro (2,5) oct-6-yl of (chloroacetyl) -carbamic acid]; preferably the interferon enhancer treatment is the hyperthermia or arginine butyrate.

Among the preferred cytostatic drugs for co-administered with interferon are included, but not limited to cyclophosphamide, cisplatin, carboplatin, carmustine (BCNU; N, N-bis (2-chloroethyl) -N-nitrosourea), methotrexate, adriamycin, α-difluoromethylomitin and 5-fluorouracil.

Cancers susceptible to this procedure include, but are not limited to, cancers that respond to parenteral administration of high doses of IFN-?, Such as malignant tumors hematologic, for example, multiple myeloma, leukemia hair cells, or chronic myelogenous leukemia, lymphomas of low grade, cutaneous T-cell lymphoma, solid tumors such as renal cell carcinoma and melanoma, the Carcinoid tumors or AIDS-associated Kaposi's sarcoma, in Particular tumors of non-viral etiology.

In the preparation of the pharmaceutical compositions used in the present invention, various vehicles and excipients for the IFN can be used, evidently for a person skilled in the art. A representative formulation technology is explained, inter alia , in Remington: The Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA, 1995 and its predecessor editions. The IFN formulation may comprise stability enhancers, such as glycine or alanine, as described in US Patent No. 4,496,537 and / or one or more transporters, such as the transport protein. For example, for human treatment, pharmaceutical grade human serum albumin is normally used, optionally together with a phosphate buffered saline solution as diluent. If the IFN excipient is human serum albumin, it may be derived from human serum or it may be of recombinant origin. Normally, when serum albumin is used, it will be homologous in origin.

The IFN can be managed through any medium that provides an IFN contact with the oromucosal cavity of the receiver. Thus, it will be clearly understood that the invention does not It is limited to any particular type of formulation. The present document describes the administration of IFN deeply in the oromucosal cavity; this can be achieved with liquids, solids or aerosols, as well as with nasal drops or nasal sprays. So, the invention includes, but is not limited to, liquid, spray, syrup, tablet, buccal tablets and nebulizer formulations. A subject matter expert will recognize that for formulations for spray or nebulizer, the particle size of the preparation It may be important, and you will be aware of proper procedures according to which the particle size can be modified.

According to one aspect of the invention, interferon It is given in a single daily dose. Alternatively, the Interferon is administered in several lower doses, distributed to over time, so that the net effect is equivalent to administration of the single largest dose. An approach to said mode of delivery is through the provision of an apparatus for sustained or controlled release adhered to or implanted in the oromucosal cavity and designed to release interferon along of time in an amount equivalent to that of a single dose high.

Among the representative formulations of Interferon for oromucosal use includes the following (all% are by weight / weight):

Tablet: 45% BP dextrose; BP gelatin 30%; BP wheat starch 11%; 5% sodium carmellose; egg albumin BPC 4%; leucine USP 3%; Propylene Glycol BP 2%; Y IFN-? 2 1 x 10 6 IU. The tablet can be used as is and let it dissolve slowly in the mouth or it can dissolve in water and keep in the mouth in contact with the oromucose, as necessary.

An interferon paste can be prepared, such and as described in US Patent No. 4,675,184, as of 45% glycerin, 2% sodium CMC, citrate buffer (pH 4.5) 25%, water distilled up to 100% and 1x 10 6 IU of IFN-? 2. Interferon Paste Can adhere to the oral mucosa.

Also, a formulation can be prepared for gargle or syrup by adding the desired amount of interferon to a commercial mouthwash formulation or a Commercial cough syrup formulation.

Within dosing intervals specific ones referred to above, the Optimal treatment in any individual case will depend on the nature of the disease in question, the stage of the disease, previous therapy, another therapy then the general state of health of the patient, the sensitivity of the subject to interferon, etc., and therefore, will be at the discretion of the doctor, taking into account those circumstances. The extension of treatment will obviously vary depending on the disease that is treating, for example, the treatment of a cancer of slow growth, such as prostate cancer, is expected to it will involve a different course of treatment than the treatment of a fast growing cancer, such as cell carcinoma hepatic.

The effective dose described herein document is one that does not generate a pathological response in the patient when administered parenterally. An answer Pathological can be acute, chronic or cumulative and can manifest by changes in blood chemistry, such as leukopenia, due to a bone marrow depression and other parameters histological In the sense used in this document, a Pathological response includes adverse side effects, such as fever, malaise or flu-like symptoms, reactions vascular, such as phlebitis and local inflammation reactions in the injection site These answers will vary considerably among the patient population depending on the variations Individuals in sensitivity to interferon. A simple analysis to identify an acceptable low dose of interferon for individual therapy is to inject the patient with the acceptable dose putative, based on considerations of age, weight, indication, progression, etc. and determine if the injection produces a response pathological as defined herein, being local irritation at the injection site the easiest criterion to establish If no adverse response is observed, you can administered the same dose via oromucosal. If there is an answer unwanted, the process is repeated at a lower dose, until it is Identify a non-pathological dose.

For many patients, the oromucosal doses are expected to be approximately the same as those known to be well tolerated and effective in existing approved parenteral protocols. Therefore, for specificity objects, an acceptable low dose of interferon will be from about 1 x 10 4 IU to about
20 x 10 6 IU of interferon per day, more preferably from about 1 x 10 4 IU to about 1 x 10 6 IU of interferon per day, as long as the dose is such that it does not induce a pathological response if administered parenterally. In one embodiment of the invention, the total dose may be administered in multiple lower doses over time, or it may be delivered continuously or pulsatically from a controlled delivery apparatus adhered to or implanted in the oromucosa.

Interferons and interferon formulations IFN- \ alpha / \ mouse

Mouse IFN-? /? (Mu IFN-? /?) Was prepared from C243-3 cell cultures induced with Newcastle disease virus (NDV) and purified as described. previously described (Tovey et al ., Proc. Soc. Exp. Biol. and Med ., 1974, 146 : 809-815). The preparation used in this study had an evaluation of 4 x 10 6 International Units (IU) / ml and a specific activity of 5 x 10 7 IU / mg protein, tested on 929 mouse cells stimulated with vesicular stomatitis virus (VSV), as previously described (Tovey et al ., Proc. Soc. Exp. Biol. and Med ., 1974, 146 : 809-815). The preparation was standardized against the international reference preparation of murine IFN-? /? Of the National Institutes of Health (NIH) (G-002-9004-541).

IFN-?-1-8 human

Recombinant human IFN-? 1-8 (Hu IFN-?; BDBB lot No. CPG 35269-1, Ciba-Geigy, Basel, Switzerland) was prepared and purified as previously described (Meister et al . , J. Gen. Virol ., 1986, 67 : 1633-1643). The preparation used in this study had an evaluation of 70 x 10 6 IU / ml on homologous human WISH cells stimulated with VSV as previously described (Tovey et al ., Nature , 1977, 267 : 455-457) and a rating of 1 x 10 6
IU / ml on heterologous mouse L929 cells. The preparation was standardized against the NIH international reference preparation of human IFN-? (G-023-901-527) and against the murine NIH IFN-? /? (G-002-9004-5411) standard. The specific activity of the IFN preparation was 2 x 10 8 IU / mg.

Recombinant Interferon- α murine

Recombinant murine interferon-α was purchased from Life Technologies Inc. The preparation used in this study (lot # HKK404) had a titre of 6 x 10 6 IU / ml and a specific activity of 6 x 10 8 } IU / mg protein, tested on mouse 929 cells stimulated with vesicular stomatitis virus (VSV) (Tovey et al ., Proc. Soc. Exp. Biol. And Med ., 1974, 146 : 406-415) .

Recombinant Murine β Interferon

Recombinant murine interferon? Was purchased from R&D Systems, Inc. The preparation used in this study (lot # 1976-01S) had a titre of 3.2 x 10 4 IU / ml and a specific activity of 8 x 10 6 IU / mg protein, tested on mouse 929 cells stimulated with vesicular stomatitis virus (VSV) (Tovey et al ., Proc. Soc. Exp. Biol. And Med ., 1974, 146 : 406-415).

Recombinant murine γ interferon

Recombinant murine interferon-γ was purchased from R&D Systems Inc. The preparation used in this study (2580-03SA) had a titre of 2 x 10 5 IV / ml and a specific activity of 1 x 10 7 IU / mg protein, tested on mouse 929 cells stimulated with vesicular stomatitis virus (VSV) (Tovey et al ., Proc. Soc. Exp. Biol. And Med ., 1974, 146 : 406-415).

All interferon preparations are simultaneously assessed in the same trial and standardized against the international reference preparation of interferon murine α / β of the National Institutes of the Health (G-002-9004-5411).

Both murine α / β interferon and recombinant murine interferons were diluted in the excipient Fe-
rimmune? before administration.

Excipient

Interferon preparations were diluted in phosphate buffered saline (PBS) containing albumin of bovine serum (BSA). Fraction V of serum albumin bovine (RIA grade; immunoglobulin free; catalog number A7888; Sigma; USA) dissolved to a final concentration of 100 µg / ml in PBS (pH 7.4) and sterilized by filtration (0.2 µ, Millex-GV, Millipore, USA).

In the experiments described herein patent, interferon preparations were diluted in a appropriate excipient. The excipient used is as follows, provided in the form of tablets (Ferimmune?, Pharma Pacific):

\ hskip0.4cm% weight / weight mg / tablet Dextrose (Glucose) BP ** \ hskip0.9cm 44.67 *** 55.84 Jelly BP ** 30.06 37.58 Starch wheat BP ** 11.31 14.14 Carmellose of sodium BP ** 4.96 6.20 Albumin of egg BPC ** 4.03 5.04 Leucine USP 3.00 3.75 Propylene glycol BP 1.88 2.35 Dextran 40 ** (as Dextran 40 injection BP) 0.06 0.08 Phosphate of sodium BP 0.03 0.04 Chloride of sodium BP 0.01 0.01 Phosphate sodium acid BP 0.01 0.01 Total 100.02 125.04 ** \, Calculated on a anhydrous base *** Derived from: \ hskip1,5cmDextrose (Glucose) BP (anhydrous) \ hskip2.14cm 44.64% BP Glucose (as Dextran injection 40 BP) 0.03%.

A single tablet was dissolved in 1.5 ml of a phosphate buffered saline, centrifuged at 16,000 g for 15 min, and then filtered under sterile conditions (0.2 µ, Millex-GV, Millipore, USA) and stored at 4 ° C before use. The excipient was prepared the day before of its use.

Interferon Delivery System

Some preliminary experiments showed that the application of 5 µl of violet glass to each window of the nose of a normal adult mouse using an Eppendorf micropipette P20 caused an almost immediate distribution of the dye throughout the entire surface of the oropharyngeal cavity. Staining of oropharyngeal cavity was still evident approximately 30 minutes after the application of the dye. Results were obtained essentially similar using IFN-? 1-8 recombinant human labeled with 125 I applied in the same way. This administration procedure is therefore used in all subsequent experiments.

For the objects of animal experiments described in the present description, it is clearly understood that the expressions "oromucosal" or "oropharyngeal" or "intranasal / oral" or "intranasal more oral" or "in / or" in relation to the administration route of the IFN they must mean the deep administration of the IFN preparation in the nasal cavity, so that it is distributed quickly in the oromucosal cavity, it is say, the mouth and throat of the recipient mammal, so that make contact with the mucosa that covers said cavity.

Friend erythroleukemia cells

The resistant IFN-? /? Clone, 3C18, or Friend erythroleukemia cells (FLC) was obtained from Dr. E. Affabris, Rome and is described in detail in Affabris et al ., 1982, ( Virology , 120 : 441-452). These cells were subsequently maintained by in vivo passages. Briefly, approximately 100 LD 50 of 3C18 cells were inoculated into DBA / 2 mice, by peritoneal injection (ip) and one week later, tumor cells were collected from the mouse peritoneum, counted and inoculated again. mice with 100 LD50 of 3C18 cells. This procedure was repeated from 60 to 100 passages. It has been shown that 3C18 cells used in the passage in vivo
60 to 100 are highly metastatic for the liver and spleen (Gresser et al ., Int. J. Cancer , 1987, 39 : 789-792). The IFN resistance phenotype was routinely confirmed by culturing cells subjected to the passages in vivo in vitro in the presence of IFN-? /? (Belardelli et al ., Int. J. Cancer , 1982, 30 : 813-820) .

The interferon compositions of the present invention are also active against clone L1210R6 of L1210 lymphoma cells isolated in our laboratory (Gresser et al ., 1974 , Interferon and cell division - interferon and cell division - IX. Interferon-resistant L1210 cells: Characteristics and Origin - interferon-resistant L1210 cells: characteristics and origin - J. Nat. Cancer Inst ., 52: 553-559) and against the EL4 transplantable tumor derived from mice inoculated with the chemical carcinogen 1-2 dimethylbenzantreine ( Gorer, PA, 1950 , Br. J. Cancer , 4: 372-381).

Animals

The mice used in the present study were obtained from a specific pathogen-free colony (IFFA CREDO, France). They were housed in an animal facility specific pathogen-free at the Institut Federatif CNRS in Villejuif, according to EEC standards.

Interferon Bioassay

Interferon was tested according to a conventional procedure. Briefly, samples (20 µl) were diluted in 80 µl
Eagle Minimum Essential Medium (MEM) (Gibco, France) containing 2% Heat-inactivated Fetal Calf Serum (FCS) (Gibco, France) and a microtiter plate (Falcon, number) was added to each well 3072) using a multichannel micropipette (Finnpipette, Labsystem, 50-300 µl). WISH cells or L929 cells (2 x 10 4 cells / well) in 100 µL of MEM containing 2% FCS were added and incubated overnight at 37 ° C in an air atmosphere with 5% of CO 2 (CO 2 incubator Form 3029). The presence of any toxicity signal in the cells was then examined using an Olympus IM GLDW inverted microscope equipped with a 10X objective. Samples that did not show a detectable toxicity were then subjected to serial double dilutions starting with an initial dilution 1:10 in a total volume of 200 µl of Eagle MEM containing 2% FCS, transferring 100 µl of material diluted with a multichannel micropipette in a microplate containing 100 µl per Eagle MEM well containing 2% FCS. Appropriate half-serial dilutions of the human NIH IFN-α reference standard (G-023-901-527) or the murine NIH IFN-α / β reference standard (G-002-9004-) were also prepared. 5411). Then, if appropriate, WISH cells or L929 cells (2 x 10 4 cells / well) in 100 µl of Eagle MEM containing 2% FCS were added to each plate and incubated overnight at 37 ° C in an air atmosphere with 5% CO2. Then, any signal of toxicity to the cell monolayers was checked and in the case of absence of any apparent toxicity, the culture was aspirated and replaced by 200 µl of Eagle MEM containing 2% FCS containing 100 TCID50 of VSV ( 2 x 10-4 VSV23 for WISH cells or 10-5 VSV23 for L929 cells). The plates were then incubated overnight at 37 ° C in an air atmosphere with 5% CO2. The viral cytopathic effect on cell monolayers was then examined using an Olympus IM ULWD inverted microscope. Interferon titers were determined from the reciprocal of the dilution, which gave 50% protection against the specific viral cytopathic effect and was expressed in international reference units / ml (IU / ml).

Example one

Anti-tumor activity of interferon-? administered by oromucosal route in mice stimulated with Friend's erythroleukemia cells

To establish if the IFN-? Administered by the intranasal route / oral (in / or) increases the survival of mice stimulated with highly metastatic FLC cells, were stimulated intravenously (iv) groups of six male DBA / 2 mice 7-8 weeks of age with 1 x 10 5 FLC on day 0.

Each group of mice was treated twice a day for 10 consecutive days using the in / or route with 104 IU of a natural mixture of murine IFN-? subtypes multiple (Mu IFN-?) in 10 µl BSA-PBS or with an equal volume of a preparation simulated IFN, which was obtained and purified in the same way that the preparation and IFN except for the omission of the inductor of virus. Simulated IFN preparation showed no IFN activity detectable when tested in parallel with the Mu preparation Purified IFN-?. The results are shown. in Table 1.

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TABLE 1

Group Treatment Day of death Average day of death \ pm BE one IFN-? Mu simulated 9, 9, 10, 10, 10, 10 9.6 ± 0.2 2 IFN-? Mu 10 4 IU 28, 31, 36,> 100,> 100 31.7 ± 2.3 ** ** Calculated only for animals dead.

Oromucosal detection of 104 IU of IFN-? Mu twice daily increased dramatically the survival time of mice that are I had injected them with FLC. In fact, 2 of 5 mice were cured indeed, since they were alive and were well 100 days after stimulation with 2 x 10 4 LD 50 of FLC, a despite stopping IFN-? treatment After only 20 days. The results of several analyzes controlled in groups of 10 adult DBA / 2 mice infected with 2 x 10 4 LD 50 of FLC and treated via oromucosal with 10 3 IU of recombinant murine IFN-? or 10 3 IU of Recombinant murine IFN-? Were Similar.

Example 2

Broader trial of anti-tumor activity of interferon-? at low dose oromucosal route in mice injected with Friend's erythroleukemia cells

To confirm the results of Example 1, a larger test was carried out. One hundred and fifty 8-week-old female DBA / 2 mice were stimulated iv with 1 x 10 5 FLC (2 x 10 4 FLC LD 50) on day 0. The mice were treated with type and dose of indicated IFN, administered by the in / or route in a volume of 10 µl twice daily
for 10 consecutive days. There were 10 mice in each treatment group. The results are shown in Table 2.

TABLE 2

Group Treatment Dose Excipient Day means of death ± SE one None - None 9.5 ± 0.2 2 IFN-? Mu simulated - BSA / PBS 9.6 ± 0.2 3 IFN-? Mu 10 4 IU BSA / PBS 29.4 ± 25.6% * 4 IFN-? Mu 10 3 IU BSA / PBS 11.6 ± 0.2 5 IFN-? Mu 10 2 IU BSA / PBS 10.3 ± 0.2 6 IFN-? 1-8 Hu 10 4 IU BSA / PBS 18.2 ± 0.8 7 IFN-? 1-8 Hu 10 3 IU BSA / PBS 13.1 ± 0.8 8 IFN-? 1-8 Hu 10 2 IU BSA / PBS 11.4 ± 0.5 IU: international reference units BSA / PBS: 100 µg / ml of BSA in PBS pH 7.4 IFN-? Mu: natural mixture of IFN-? subtypes murine IFN-? Hu: unique isotype of Recombinant human IFN-? *: some of this animals group were still alive 100 days after treatment

IFN-? Administered using the in / or route shows a marked activity anti-tumor in mice stimulated iv with FLC. From In fact, some mice treated with IFN can be considered cured, since they were still alive more than 100 days after the inoculation of 100,000 FLC in a system in which 4 to 5 cells FLC are enough to kill the animal.

Pure preparations of a single subspecies of recombinant IFN-?, IFN-? 1-8, of a mixture multi-type natural IFN-α, from IFN-? Mu, from IFN-? recombinant murine and murine IFN-? recombinant show a marked activity anti-tumor in this model if administered through the in / or path, which strongly suggests that the activity anti-tumor observed from IFN preparations administered by oromucosal route is, in fact, due to the component IFN of such preparations.

Example 3

Effect of the interferon administration route on the anti-tumor activity

The effect of IFN administered in / or was compared with that of IFN administered by conventional routes. Be stimulated eight week old iv female DBA / 2 mice with 1 x 10 5 FLC (2 x 10 4 FLC LD 50) on day 0. Mice they were treated twice a day for 10 consecutive days with 10 4 IU of IFN-? Mu (the optimal dose is as determined in Example 2) administered by the indicated route. There were 6 mice in each treatment group and in each case, the excipient for IFN-? Mu was BSA in PBS. The results are shown in Table 3.

TABLE 3

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Group Treatment Route Day means of death ± SE one None - 9.6 ± 0.2 2 IFN-? Mu Intranasal / oral 30 ± 25.85% * 3 IFN-? Mu oral 18.5 ± 2.0 4 IFN-? Mu gastric 13.5 ± 1.3 5 IFN-? Mu subcutaneous 23.5 ± 1.6 6 IFN-? Mu intramuscular 23.7 ± 1.8 7 IFN-? Mu intravenous 25.0 ± 1.2 8 IFN-? Mu intraperitoneal 26.7 ± 4.1 UI: units of international reference BSA / PBS: 100 µg / ml of BSA in PBS pH 7.4 *:  \ begin {minipage} [t] {150mm} some of the animals in this group they were still alive 100 days after the inoculation of the FLC \ end {minipage} cells

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The oromucosal administration route (or in / or) It is at least as effective as the parenteral routes used commonly, such as iv, intramuscular injection (im) and subcutaneous (sc), if not more effective. The in / or route was in fact as effective as ip injection, which is considered the most effective in mice stimulated iv with FLC. In contrast to what above, the administration of the same dose of IFN directly in mouth proved to be less effective than combined administration intranasal and oral, while the introduction of IFN directly in the stomach of animals through a tube was considerably less effective.

Example 4

Effect of oromucosal interferon on the expression of cellular proteins

It is known that IFN-? induces the expression of various cellular proteins after binding of the protein to its cell surface receptor. Be think that these proteins provide a useful marker of the IFN action.

We evaluate the effect of IFN-? Administered via the in / or path over the expression of three IFN-induced proteins, the MHC antigens of class I, the Ly 6A / E antigen and the 2'-5'-oligoadenylate synthetase.

The treatment of mice DBA-2 (H-2K d) with up to 20,000 IFN-? Mu UI via the in / or no path significantly increased the expression of H-2-K d in lymphocytes, monocytes or granulocytes of peripheral blood under conditions in which an amount as small as 20 IU of IFN-? Mu administered ip increased sharply antigen expression H-2-K d both on monocytes as on peripheral blood granulocytes. From In fact, the expression of monocytes was suppressed slightly.

Similarly, treatment of mice with up to 20,000 IU of IFN-α via the in / or pathway did not have a significant effect on the expression of Ly6 A / E antigens, the expression of which increases markedly on the surface of a variety of lymphoid cells after parenteral treatment with type I IFN (Dumont et al ., J. Immunol ., 1986, 137 : 201-210). Similar results were obtained with 200 or 200,000 IU of IFN-? Mu or IFN-? 1-8 Hu via the in / or route.

Treatment of Swiss or DBA / 2 mice with a amount so small cone 20 IU of IFN-? Mu injected ip resulted in a marked increase in activity 2'-5'-oligoadenylate synthetase both in peripheral mononuclear cells as in splenocytes. In contrast to the above, in the same experiment, the treatment of mice with up to 20,000 IU of IFN-? Mu via the in / or path did not increase significantly the expression of the activity 2'-5'-oligoadenylate synthetase. In addition, treatment with 200 or 200,000 IU of IFN-? Mu or IFN-? 1-8 Hu via the in / or route did not have an effect significant about the activity 2'-5'-oligoadenylate synthetase in any of the times analyzed up to 10 days after start of treatment with IFN.

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Example 5

Bioavailability of interferon after administration oromucosal

To examine the bioavailability and IFN pharmacokinetics, mice that had the relationship were treated in drug-blood volume more favorable for these studies, with a single high dose of IFN-? Recombinant labeled until highest specific radioactivity possible with125I.

A pure preparation of 70 x 10 6 IU of IFN-? 1-8 Hu was dissolved in 1.4 ml of PBS and iodinated as described in Mogensen et al . ( Int. J. Cancer ., 1981, 28 : 575-582) using a modification of the chloramine-T procedure described by Hunter and Greenwood ( Nature , 1962, 194 : 495-496).

IFN-? 1-8 Hu marked with125I (lot no. CGP35269-1) showed a biological activity of 2 x 10 7 IU / ml when tested on human WISH cells stimulated with VSV and 1 x 10 6 IU / ml when tested on L929 mouse cells stimulated with VSV.

They were injected iv, ip or treated in / or mice Swiss female six to seven weeks old with 2 x 10 7 IU, equivalent to 1 x 10 6 murine IU, of IFN-? 1-8 Hu125I (1.0369 x 10 7 cpm / mouse). At the indicated times, it three mice were sacrificed per group, blood was drawn and determined the volume Kidney, liver, lung, spleen and stomach / esophagus, dried and weighed with an accuracy of ± 1.0 \ mug. The radioactivity of each sample was determined individually using a gamma counter. Then the whole blood by centrifugation (800 g x 10 min., 4 ° C), is The serum was collected, counted and frozen at -80 ° C. Then it was analyzed IFN content in serum using a standard bioassay on WISH human cells and on L929 mouse cells, as has been described above. Then the radioactive material present in the serum samples was isolated by affinity immunoprecipitation and was analyzed by SDS-PAGE.

Radioactivity levels were detected very high (> 2 x 10 6 cpm / ml) in the peripheral blood of the animals 5 minutes after injection of 1.0369 x 10 7 cpm / IFN-? 1-8 Hu mouse labeled with 125 I by iv bolus. So, the amount of radioactivity present in whole blood declined progressively at 15 and 30 minutes. Radioactivity levels detected in the peripheral blood of animals 5 minutes later of the ip injection of 1.0369 x 10 7 cpm of IFN-? 1-8 Hu125I was approximately twenty times lower than the levels detected after an iv bolus injection. Radioactivity levels then progressively increased to 15 and 30 minutes after the injection. The radioactivity levels detected in the blood of the animals 5, 10 or 15 minutes after the administration in / or of IFN-? 1-8125 I were significantly lower than those detected at a time determined after ip injection of the same amount of IFN radiolabelled By the three routes of administration, they were detected higher levels of radioactivity in the serum than in the blood complete after in / or administration of IFN-? 1-8 marked with 125 I. The lowest levels of radioactivity detected per ml of whole blood compared to the same serum volume they reflect an effectively larger volume of serum counted after the separation of the cellular component from whole blood.

The presence of IFN was analyzed biologically active in serum samples of all mice in the study using a standard bioassay, as described more above, and showed easily detectable levels of IFN biologically active in the serum of all animals that are had injected either iv or ip with IFN-? 1-8 Hu125 I at all times analyzed. In In contrast to the above, biologically active IFN was not detected in the serum of any of the animals at any time analyzed after the in / or administration of IFN, despite the presence of relatively high levels of radioactivity in the serum of said animals.

To determine whether the radioactive material detected in the serum of animals treated with IFN-? 1-8 Hu125I in fact represents the native IFN, the samples are immunoprecipitated with random A-G protein, to precipitate the immunoglobulins present in the samples, treated with an affinity purified polyclonal antibody anti-IFN-? and they were again immunoprecipitate Then the samples were subjected to a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described above.

SDS-PAGE analysis of the radioactive material in the serum after iv or ip injection of IFN-? 1-8 Hu125 I revealed a single homogeneous band that migrated with an electrophoretic mobility identical to that corresponding without injection of IFN-? 1-8 Hu125I. The apparent molecular weight of the material was estimated at approximately 20,000 Daltons, which corresponds exactly to the molecular weight of the native IFN-? 1-8 Hu. In contrast to the above, none of the mouse serum samples treated in / or with IFN-? 1-8125I contained material with an apparent molecular weight similar to that of the native IFN, even when an amount was charged identical radioactive material in each
gel.

The tissue distribution of the material Radiolabeling revealed very high levels of radioactivity in kidneys, high levels in the liver, lung and spleen of the animals 5 minutes after iv injection of IFN-? 1-8125 I. Be discovered then that the level of radioactivity present in each one of these four organs decreased progressively at 15 and 30 minutes In contrast to the above, the level of radioactivity in the stomach progressively increased at 15 and 30 minutes, until reach a level comparable to that present in animal serum 30 minutes after an iv bolus injection.

The administration of IFN-? 1-8125 I by ip injection resulted in radioactivity spikes in all tissues examined at 15 minutes, followed by a decline to 30 minutes Similarly, the in / or administration of IFN-? 1-8 Hu125I gave as a result radioactivity peaks in all tissues studied after 15 minutes, with some decline in Radioactivity levels present at 30 minutes. The levels of radioactivity present in the stomach / esophagus were of an order of magnitude greater than those detected in any other organ after of the in / or administration of IFN-? 1-8 marked with125I and was markedly greater that the levels present in these tissues after parenteral administration of the same amount of IFN-? 1-8 Hu radiolabeled both by route iv and by route ip.

Example 6

Pharmacokinetics of interferon after administration intranasal / oral

For the precise determination of the IFN-? 1-8 Pharmacokinetics Hu, iv, ip or in / or mice were treated with 1.0369 x 10 7 cpm / IFN-? 1-8 Hu mouse labeled with125I and the levels of radioactivity present in whole blood and serum at a series of times over a period of 24 hours.

The pharmacokinetic profile of IFN-? 1-8 Hu labeled with 125 I present in the blood of the mice after an iv bolus injection closely followed a logarithmic elimination curve. The above was in agreement with the results of a previous study conducted in mice using a closely related molecule, recombinant human α / A (Bgl) (Bohoslawed et al ., J. IFN Res ., 1986, 6 : 207- 213). The amount of bioavailable material, calculated from the area under the concentration versus time curve, was also similar to that of human α A / D. An elimination curve with biphasic time consumption was observed after an IV bolus injection of IFN-? 1-8125 I, which is characteristic of the substances that are removed through the kidneys, according to the results of Example 4. The pharmacokinetics of IFN-? 1-8 labeled with125I after ip injection closely resembled that previously described for IFNs administered im.

They were present in everyone's serum animals easily detectable levels of biologically IFN active after an iv bolus injection or an ip injection of IFN-? 1-8 marked with 125 I.

Friend's erythroleukemia model constitutes a severe preclinical analysis of activity anti-tumor, since FLC is highly malignant and It carries liver and spleen metastases when injected iv. In fact, the results obtained using this model were the basis for the adoption of parenteral injection of IFN-? For the treatment of cancers humans. So, in all the experiments carried out in this study, all untreated animals and animals treated with Control preparations died 10 to 11 days later. The Injection of only 4 or 5 FLC cells would kill a mouse if not It provides no treatment. In contrast to the above, some animals treated with murine RFN-? through the oromucosal route they still lived more than 100 days later of inoculation of 10 5 FLC and can be considered cured.

In fact, judging from previous work, IFN-? Administered by the oromucosal route seems to be more effective than cyclophosphamide, 5-fluorouracil or methotrexate administered parenterally, increasing the survival time only a few days in animals injected with FLC (Gresser et al ., J. Natl. Cancer Inst ., 1988, 80 : 126-131). Other drugs, for example cisplatin, vincristine, doxorubicin, bleomycin or etoposide are not effective against this tumor (Gresser et al ., J. Natl. Cancer Inst ., 1988, 80 : 126-131).

Similarly, IFN-? Administered by the oromucosal route appears to be more effective against FLC than other cytokines such as IL-1?, IL-2 and systemically administered TNF-?, Which exhibit very little activity in this
model.

Previous work has shown that IFN administered parenterally is one of the most active anti-tumor drugs in this model and that IFN therapy is effective even when initiated after tumor metastases are already present in the liver (Gresser et al ., Int. J. Cancer , 1987, 39 : 789-792). The present results show that the administration of IFN through the oromucosal route is equal or even more effective.

Daily injections of IFN-? Given together with a single dose of cyclophosphamide markedly increased the survival of AKR mice that had lymphoma compared to animals treated with any isolated agent, if therapy began before the diagnosis of lymphoma (Gresser et al . , Eur. J. Cancer , 1978, 14 : 97-99). Successful combination therapy using IFN-? Z? And BCNU, cis-DDP (cisplatin), methotrexate, adriamycin and? -Difluoromethyl ornithine has also been described in several pre-clinical animal tumor models. It has also been described that combination therapy with 5-fluorouracil (5-FU) and IFN is beneficial in the treatment of metastatic colon cancer in humans (Ernstoff et al ., Journal of Clinical Oncology , 1989, 7 : 1764-1765 ). However, there are other studies that have described a decreased anti-tumor activity if IFN therapy was combined with the use of cyclophosphamide (Marquet et al ., Int. J. Cancer , 1983, 31 : 223-226; Lee et al . , Biochem. Pharmacol ., 1984, 33 : 4339-3443), adriamycin (Blackwill et al ., Cancer Res ., 1984, 44 : 904-908) or 5-FU (Marquet et al ., 1985, 109 : 156- 158), that is, precisely the same drugs that have been shown to have a beneficial effect when used in combination with parenteral IFN therapy. Combinations between IFN and other chemotherapy agents can be easily analyzed using the procedures described in the present invention.

Combined interleukin-1 (IL-1) and IFN-? /? Therapy results in a synergistic anti-tumor effect in mice that have been injected with FLC (Belardelli et al ., Int. J. Cancer , 1991, 49 : 274-278). The same treatment also exerts a marked anti-tumor effect against a metastatic variant (p11-R-Eb) of Eb lymphoma, against which any agent alone is not effective (Gabriele et al ., Invasion Metastasis , 1993, 13 : 147- 162). Of all the cytokines analyzed, IL-1 was found to be the most effective if combined with parenteral Type I IFN therapy.

Combined therapy with the AGM-1470 angiogenesis inhibitor [ester (3R- (3α, 4α (2R *, 3R *), 5β, 6β)) - 5-methoxy-4- ( 2-Methyl-3- (3-methoxy-2-butenyl) oxyranyl) -1-oxaspiro (2.5) oct-6-yl (chloroacetyl) -carbamic acid] administered together with IFN-? /? Resulted in a markedly increased anti-tumor effect compared to that observed with any agent alone (Brem et al ., J. Pediatric Surgery , 1993, 28 : 1253-1257).

Hyperthermia has been shown to increase the anti-tumor action of IFN-? /? Against Lewis lung carcinoma (Yerushalmi et al ., Proc. Soc. Exp. Biol. Med ., 1982, 169 : 413- 415). Arginine butyrate has also been shown to potentiate the anti-tumor action of IFN-? (Chany and Cerutti, Int. J. Cancer , 1982, 30 : 489-493).

The comparison of the degree of protection obtained when a given type and dose of IFN is given using the oromucosal route compared to the results obtained following systemic administration (ip injection) showed that parenteral administration of IFN in some cases was marginally more effective and in other cases no more effective than Oromucosal administration.

The results of the pilot study with biomarker showed quite clearly that none of the three Biomarkers analyzed (MHC class I antigen, Ly6 A / E antigen and 2'-5'-oligoadenylate activity synthetase) adequately reflects the very marked activity antitumor that exhibits administered IFN-? through the oromucosal route.

The contrast between the very marked is highlighted increase in the expression of the IFN-induced proteins observed in all experiments carried out after the ip injection of an amount as small as 20 IU of IFN-? And the absence of any effect detectable after administration of up to 20,000 IU of IFN-? Via the oromucosal route.

Although we cannot exclude the possibility of observe at an earlier or intermediate time an effect on one or another of the biomarkers, this seems to be unlikely, since IFN acts on the transcription of the genes that encode these proteins and therefore it is not expected to observe an effect on any of said biomarkers until several hours after IFN treatment.

Again, although we cannot exclude the possibility of observing a systemic effect on one or the other of the numerous IFN-induced proteins after treatment with IFN-? Via the oromucosal path, this it seems unlikely, since this would imply regulation differential on the expression of certain IFN-induced genes. However, it is completely possible that it can be observed locally an effect on an IF biomarker, for example in nasal lymphocytes, after administration of IFN-? Via the oromucosal route.

According to the absence of an effect detectable on the biomarkers studied, no consistent effect on any of the chemical parameters Hematologic or blood monitored during IFN therapy oromucosal, even in animals treated with up to 20,000 IU of IFN-?

Easily detectable levels of radiolabeled material were found both in whole blood and in the serum of the animals after the oromucosal administration of IFN-? 1-8 labeled with125I. These results contrast with the results of previous studies, which were not able to detect IFN in the animals' serum even after oral administration of large amounts of unlabeled IFN. However, the radioactive material detected in both whole blood and serum after oromucosal administration was biologically inactive. In addition, the results of the SDS-PAGE analysis showed that this material was of low molecular weight and most likely reflected the absorption of degradation products after digestion of IFN in the stomach and small intestine. Analysis of the tissue distribution of radiolabelled material after oromucosal administration revealed markedly higher levels of radioactivity in the stomach than in any of the other organs analyzed. Our results show quite clearly that although biologically active IFN was not absorbed after oromucosal administration, however this treatment exerts a statistically significant antitumor activity in vivo .

Without trying to limit yourself to any mechanism proposed for the observed beneficial effect, our results suggest that IFN administered via oromucosal exerts its effect against cancer cells via a novel mechanism not currently defined, which does not imply a direct action of the IFN exogenously administered or induction of endogenous IFN. This is left backed by the absence of detectable levels of circulating IFN or of the three biomarkers analyzed, it seems that this mechanism can act at least partially by stimulating the abundant lymphoid tissue surrounding the nasopharyngeal cavities and the oral cavity. Since we have shown that the oromucosal IFN is at least effective comparable to the administered IFN systemically, our results provide strong support for the administration of IFN through the oromucosal route in the cancer treatment This can have important implications. for the clinical use of the IFN.

Claims (15)

1. Use of human interferon for preparation of a medicine in the treatment of cancer in a human by administration by contact with the coating mucosa of the mouth and throat of the human recipient from 10 4 IU up to 20 x 10 6 IU of interferon per day. 2. Use according to claim 1, in the which cancer is a multiple myeloma, a cell leukemia hairy, chronic myelogenous leukemia, low grade lymphoma, a cutaneous T-cell lymphoma, a carcinoid tumor, a cancer cervical sarcomas, including Kaposi's sarcoma, the tumor of kidney, carcinomas, including renal cell carcinoma, hepatic cell carcinoma, nasopharyngeal carcinoma, hematological malignancies, colorectal cancer, the glioblastoma, laryngeal papilloma, lung cancer, cancer colon, malignant melanoma or brain tumors, including the malignant brain tumor. 3. Use according to claim 1 or 2, in which medication comprises a single dose of interferon. 4. Use according to claim 1 or 2, in which the medicine comprises a set of smaller doses of interferon sufficient to obtain a response equivalent to that of a single dose, without inducing a pathological response. 5. Use according to any of the claims 1-4, wherein the medicament provides a continuous administration of a dose of interferon during a enough time to get an answer antineoplastic equivalent to that of a single dose, without inducing a pathological response 6. Use according to any of the claims 1-5, wherein the medicament comprises another cytokine or an interferon inducer. 7. Use according to any of the claims 1 to 6, wherein the medicament is adapted to the administration of 1 x 10 4 to 1 x 10 6 IU of interferon by day. 8. Use according to any of the claims 1 to 7, wherein the medicament includes another therapeutic agent for simultaneous, separate or sequential. 9. Use according to claim 8, in the which therapeutic agent is selected from the group consisting in cytostatic agents, anti-cancer agents and anti-angiogenic agents 10. Use according to any of the claims 1 to 9, wherein the interferon is an interferon of Type I. 11. Use according to claim 10, wherein the interferon is selected from the group consisting of
IFN-?, IFN-?, IFN-?, IFN consensus and mixtures thereof. 12. Use according to claim 11, in which Type I interferon is IFN-?. 13. Use according to any of the claims 1-9 wherein the interferon is an interferon of Type II 14. Use according to claim 13, in which Type II interferon is IFN- γ. 15. Use according to claim 10 or 13, in which interferon is a recombinant material.